Genomic DNA from mouse and rat have been analyzed by a two-dimensional technique which permits greatly improved resolution of DNA segments defined on the basis of sites for restriction enzymes. After first degesting the DNA with one or more restriction enzymes, the generated fragments are separated by electrophoresis in a gel column of purified agarose which contains the second enzyme. The conditions of the first dimension run are chosen so that the second enzyme is immobilized and inactive during the run, while permitting the DNA fragments to migrate according to their length. After the first electrophoresis, the gel column is incubated under conditions to promote the activity of the second enzyme. Cleavage having been completed, the gel column is enbedded in a slab of agarose for a second electrophoretic run perpendicular to the first. Some sequences in the mouse and rat genome are sufficiently abundant to produce discrete spots, streaks, and secondary diagonals when the gels are stained; other sequences require the use of specific probes for identification. The secondary diagonals and streaks, generated by the second restriction enzyme, connect specific sequences that are more heterogeneously defined by the first enzyme.